A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical derived tissue specimens
Abstract Systematic quantification of phosphoprotein within cell signaling networks in solid tissues remains challenging and precise quantification in large scale samples has great potential for biomarker identification and validation. We developed a reverse phase protein array (RPPA) based phosphor...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2022-12-01
|
| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-26715-9 |
| _version_ | 1797973668596285440 |
|---|---|
| author | Nan Wang Li Zhang Qi Ying Zhentao Song Aiping Lu Achim Treumann Zhaojian Liu Tao Sun Zhiyong Ding |
| author_facet | Nan Wang Li Zhang Qi Ying Zhentao Song Aiping Lu Achim Treumann Zhaojian Liu Tao Sun Zhiyong Ding |
| author_sort | Nan Wang |
| collection | DOAJ |
| description | Abstract Systematic quantification of phosphoprotein within cell signaling networks in solid tissues remains challenging and precise quantification in large scale samples has great potential for biomarker identification and validation. We developed a reverse phase protein array (RPPA) based phosphor-antibody characterization approach by taking advantage of the lysis buffer compatible with alkaline phosphatase (AP) treatment that differs from the conventional RPPA antibody validation procedure and applied it onto fresh frozen (FF) and formalin-fixed and paraffin-embedded tissue (FFPE) to test its applicability. By screening 106 phospho-antibodies using RPPA, we demonstrated that AP treatment could serve as an independent factor to be adopted for rapid phospho-antibody selection. We also showed desirable reproducibility and specificity in clincical specimens indicating its potential for tissue-based phospho-protein profiling. Of further clinical significance, using the same approach, based on melanoma and lung cancer FFPE samples, we showed great interexperimental reproducibility and significant correlation with pathological markers in both tissues generating meaningful data that match clinical features. Our findings set a benchmark of an efficient workflow for phospho-antibody characterization that is compatible with high-plex clinical proteomics in precison oncology. |
| first_indexed | 2024-04-11T04:07:57Z |
| format | Article |
| id | doaj.art-eb6ec0a543774594b689a466d84493f1 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-11T04:07:57Z |
| publishDate | 2022-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-eb6ec0a543774594b689a466d84493f12023-01-01T12:19:03ZengNature PortfolioScientific Reports2045-23222022-12-0112111310.1038/s41598-022-26715-9A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical derived tissue specimensNan Wang0Li Zhang1Qi Ying2Zhentao Song3Aiping Lu4Achim Treumann5Zhaojian Liu6Tao Sun7Zhiyong Ding8Mills Institute for Personalized Cancer Care, Fynn BiotechnologiesDepartment of Pathology, Beijing Cancer HospitalMills Institute for Personalized Cancer Care, Fynn BiotechnologiesMills Institute for Personalized Cancer Care, Fynn BiotechnologiesDepartment of Pathology, Beijing Cancer HospitalNewcastle University Protein and Proteome Analysis, Newcastle UniversityDepartment of Cell Biology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong UniversityDepartment of Haematology, Qilu Hospital, Cheeloo College of Medicine, Shandong UniversityMills Institute for Personalized Cancer Care, Fynn BiotechnologiesAbstract Systematic quantification of phosphoprotein within cell signaling networks in solid tissues remains challenging and precise quantification in large scale samples has great potential for biomarker identification and validation. We developed a reverse phase protein array (RPPA) based phosphor-antibody characterization approach by taking advantage of the lysis buffer compatible with alkaline phosphatase (AP) treatment that differs from the conventional RPPA antibody validation procedure and applied it onto fresh frozen (FF) and formalin-fixed and paraffin-embedded tissue (FFPE) to test its applicability. By screening 106 phospho-antibodies using RPPA, we demonstrated that AP treatment could serve as an independent factor to be adopted for rapid phospho-antibody selection. We also showed desirable reproducibility and specificity in clincical specimens indicating its potential for tissue-based phospho-protein profiling. Of further clinical significance, using the same approach, based on melanoma and lung cancer FFPE samples, we showed great interexperimental reproducibility and significant correlation with pathological markers in both tissues generating meaningful data that match clinical features. Our findings set a benchmark of an efficient workflow for phospho-antibody characterization that is compatible with high-plex clinical proteomics in precison oncology.https://doi.org/10.1038/s41598-022-26715-9 |
| spellingShingle | Nan Wang Li Zhang Qi Ying Zhentao Song Aiping Lu Achim Treumann Zhaojian Liu Tao Sun Zhiyong Ding A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical derived tissue specimens Scientific Reports |
| title | A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical derived tissue specimens |
| title_full | A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical derived tissue specimens |
| title_fullStr | A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical derived tissue specimens |
| title_full_unstemmed | A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical derived tissue specimens |
| title_short | A reverse phase protein array based phospho-antibody characterization approach and its applicability for clinical derived tissue specimens |
| title_sort | reverse phase protein array based phospho antibody characterization approach and its applicability for clinical derived tissue specimens |
| url | https://doi.org/10.1038/s41598-022-26715-9 |
| work_keys_str_mv | AT nanwang areversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT lizhang areversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT qiying areversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT zhentaosong areversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT aipinglu areversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT achimtreumann areversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT zhaojianliu areversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT taosun areversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT zhiyongding areversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT nanwang reversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT lizhang reversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT qiying reversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT zhentaosong reversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT aipinglu reversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT achimtreumann reversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT zhaojianliu reversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT taosun reversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens AT zhiyongding reversephaseproteinarraybasedphosphoantibodycharacterizationapproachanditsapplicabilityforclinicalderivedtissuespecimens |